Computational protocol: The Effect of Central Loops in miRNA:MRE Duplexes on the Efficiency of miRNA-Mediated Gene Regulation

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Protocol publication

[…] We defined a relatively relaxed criterion for the seed region of the MRE so that more possible miRNA targets could be included in the primary screening. A flexible miRNA “seed window” (nt1–6, 2–7 and 3–8 counted from the 5′ end of the miRNA) was used to scan the 3′-UTRs of a gene in the order of 5′-3′ for potential target sites. In the seed windows, we searched for a perfectly Watson-Crick–base-paired stretch of 6 nt but tolerated one G-U wobble.After the seed regions were identified, the upstream flanking region of the seed and the seed region itself was extracted for Dynamic programming. The miRNA:mRNA duplex and the MRE sequences of the VEGF 3′-UTR were identified with Dynamic hybridization. The distal sequences in the miRNA:mRNA duplex were scored with revised Gotoh's and Marks' methods , . A score of +5 was given to G:C and A:T pairs, +2 to G:U wobble pairs, and −3 to mismatch pairs, with the gap-open and gap elongation parameters set to −8.0 and −2.0 respectively. The final scores of the miRNA:mRNA duplexes were the sum of single residue-pair match scores . Then the sequences of MREs identified by Dynamic hybridization in the VEGF 3′-UTR and potential miRNA were used as input in RNAcofold to predict the RNA secondary structures of the miRNA:mRNA duplexes. If the length of the MRE identified by Dynamic programming was not long enough to form the miRNA:mRNA duplexes calculated by RNAcofold, the duplex would be simulated again using the same mRNA sequence. This time, the sequence used would begin further upstream, such that the region used was 40nt in length including the seed region.RNAcofold most lately released in the Vienna RNA Package 1.5 beta version (www.tbi.univie.ac.at/ivo/RNA/) was incorporated into the FindTar version 1.0 algorithm. Sequences of MREs in the VEGF 3′-UTR and potential miRNA were input to precisely predict the RNA secondary structures of the miRNA:mRNA duplexes by calculating the minimum free energy (ΔG) of the whole miRNA:mRNA duplex. […]

Pipeline specifications

Software tools RNAcofold, ViennaRNA
Application RNA structure analysis
Organisms Homo sapiens